The kynurenine pathway has received increasing attention as its connection to inflammation, the immune system and neurological conditions has become more apparent. It is the primary route for tryptophan catabolism in the liver and the starting point for the synthesis of nicotinamide adenine dinucleotide in mammals. Dysregulation or overactivation of this pathway can lead to immune system activation and accumulation of potentially neurotoxic compounds. These aspects make the kynurenine pathway a promising target for therapeutic development to treat inflammation and disease with neurological aspects, especially in cancer patients undergoing chemotherapy.

Proper visualization of scientific data is important for understanding spatial relationships. Particularly in the field of structural biology, where researchers seek to gain an understanding of the structure and function of biological macromolecules, it is important to have access to visualization programs which are fast, flexible, and customizable. We present KiNG, a Java program for visualizing scientific data, with a focus on macromolecular visualization. KiNG uses the kinemage graphics format, which is tuned for macromolecular structures, but is also ideal for many other kinds of spatially embedded information. KiNG is written in cross‐platform, open‐source Java code, and can be extended by end users through simple or elaborate “plug‐in” modules. Here, we present three such applications of KiNG to problems in structural biology (protein backbone rebuilding), bioinformatics of high‐dimensional data (e.g., protein sidechain chi angles), and classroom education (molecular illustration). KiNG is a mature platform for rapidly creating and capitalizing on scientific visualizations. As a research tool, it is invaluable as a test bed for new methods of visualizing scientific data and information. It is also a powerful presentation tool, whether for structure browsing, teaching, direct 3D display on the web, or as a method for creating pictures and videos for publications. KiNG is freely available for download at http://kinemage.biochem.duke.edu.

CYP121, the cytochrome P450 enzyme in Mycobacterium tuberculosis that catalyzes a single intramolecular C–C cross-linking reaction in the biosynthesis of mycocyclosin, is crucial for the viability of this pathogen. This C–C coupling reaction represents an expansion of the activities carried out by P450 enzymes distinct from oxygen insertion. Although the traditional mechanism for P450 enzymes has been well studied, it is unclear whether CYP121 follows the general P450 mechanism or uses a different catalytic strategy for generating an iron-bound oxidant. To gain mechanistic insight into the CYP121-catalyzed reaction, we tested the peroxide shunt pathway by using rapid kinetic techniques to monitor the enzyme activity with its substrate dicyclotyrosine (cYY) and observed the formation of the cross-linked product mycocyclosin by LC-MS. In stopped-flow experiments, we observed that cYY binding to CYP121 proceeds in a two-step process, and EPR spectroscopy indicates that the binding induces active site reorganization and uniformity. Using rapid freeze-quenching EPR, we observed the formation of a high-spin intermediate upon the addition of peracetic acid to the enzyme–substrate complex. This intermediate exhibits a high-spin (S = 5/2) signal with g values of 2.00, 5.77, and 6.87. Likewise, iodosylbenzene could also produce mycocyclosin, implicating compound I as the initial oxidizing species. Moreover, we also demonstrated that CYP121 performs a standard peroxidase type of reaction by observing substrate-based radicals. On the basis of these results, we propose plausible free radical–based mechanisms for the C–C bond coupling reaction.

Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a non-heme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2. Here, using EPR, Mössbauer, and UV-Vis spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate to the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed a substrate-mediated reduction of the ferric to the ferrous oxidation state at the iron center. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases.

The kynurenine pathway is the primary route for L-tryptophan degradation in mammals. Intermediates and side products of this pathway are involved in immune response and neurodegenerative diseases. This makes the study of enzymes, especially those from mammalian sources, of the kynurenine pathway worthwhile. Recent studies on a bacterial version of an enzyme of this pathway, 2-aminomuconate semialdehyde (2-AMS) dehydrogenase (AMSDH), have provided a detailed understanding of the catalytic mechanism and identified residues conserved for muconate semialdehyde recognition and activation. Findings from the bacterial enzyme have prompted the reconsideration of the function of a previously identified human aldehyde dehydrogenase, ALDH8A1 (or ALDH12), which was annotated as a retinal dehydrogenase based on its ability to preferentially oxidize 9-cis-retinal over trans-retinal. Here, we provide compelling bioinformatics and experimental evidence that human ALDH8A1 should be reassigned to the missing 2-AMS dehydrogenase of the kynurenine metabolic pathway. For the first time, the product of the semialdehyde oxidation by AMSDH is also revealed by NMR and high-resolution MS. We found that ALDH8A1 catalyzes the NAD+ -dependent oxidation of 2- AMS with a catalytic efficiency equivalent to that of AMSDH from the bacterium Pseudomonas fluorescens. Substitution of active-site residues required for substrate recognition, binding, and isomerization in the bacterial enzyme resulted in human ALDH8A1 variants with 160-fold increased K_m or no detectable activity. In conclusion, this molecular study establishes an additional enzymatic step in an important human pathway for tryptophan catabolism.